System, method and computer program for non-binary sequence comparison

ABSTRACT

A system and method for performing non-binary comparison of biological sequences includes a new measure ω 0 , which is a non-binary counting measure that is used in a stand alone module called VaSSA-1. This measure obtains substantially more information about sequences and comparisons between them than is gathered by conventional bioinformatics techniques.

This application is a continuation application of U.S. Ser. No.11/378,284, filed Mar. 20, 2006, which claims priority from U.S.Provisional Application Ser. No. 60/662,943, filed Mar. 18, 2005. Theentirety of all of the aforementioned applications is incorporatedherein by reference.

FIELD

The present invention relates generally to bioinformatics, and moreparticularly to methods for determining the degree of similarity anddifference between genetic sequences.

BACKGROUND

DNA sequences of entire genomes of different species are beingdetermined at a rapid rate. It is incumbent on the bioinformaticscommunity to understand these genomic structural variations andfunctions. Also, some finished versions of genome data contain gapswhere data could not be acquired. These drafts of various genomicsequence data may consist of pieces of data whose relative order andorientation are difficult to determine. Dealing with such incompletedata places new demands upon integrative systems tools, particularlywhen two or more genomes are being compared. The bioinformaticscommunity needs to be able to handle gaps more effectively.

In conventional approaches, handling comparisons across genomes is amajor problem. For extremely similar sequences, there exist so called“greedy” alignment methods that compute optimal alignments. Thesealgorithms allow gaps in the alignments and are extremely efficient, butwork well only for very simple alignment scoring schemes. For richerscores (involved in large stretches of a single genome and comparingmultiple genomes), these greedy methods lose their efficiency edge overdynamic programming.

Conventional alignment methods for three or more sequences are almostentirely geared toward comparison of protein sequences based on putativecodons, sets of three nucleic acid bases encoding a single amino acid.This may be due to the fact that few examples exist of genomic sequencedata from several similar species. Also, sequence comparisons andhomology analyses are done on a binary basis. This conservescomputational resources, but ignores biochemical information.

There is a need for an improved solution that overcomes shortcomings ofconventional sequence alignment similarity and gene sequence comparisontools.

SUMMARY

A system for sequence analysis comprises an analysis module adapted tocalculate a non-binary similarity score between a first nucleotidesequence and a second nucleotide sequence; a file management module; anda plot module.

In one embodiment, the system further comprises a report module, a useroptions module and/or a user help module.

In another embodiment, the file management module comprises a loadsequences module, adapted to load at least one sequence file; a flushactive sequence module, adapted to flush a sequence file from a memory;and a flush loaded sequence module, adapted to flush a loaded sequencefile from the memory.

In another embodiment, the load sequence module comprises a loadedsequence display module, adapted to generate and display a summaryreport notebook page when a sequence is loaded, wherein the summaryreport notebook page is adapted to display a sequence file name and anumber of sequences.

In another embodiment, the report module is adapted to generate anddisplay a sequence summary, a listing of the contents of each loadedsequence, and/or statistical information about each loaded sequence.

In another embodiment, the analysis module comprises an align sequencesmodule adapted to align a target sequence to a base sequence and todisplay an alignment report; an ω₀ module adapted to calculate an ω₀score for a sequence and to display the ω₀ score; a query repeat moduleadapted to locate multiple occurrences of the target sequence in thebase sequence and to display the multiple occurrences; a query omegarepeats module adapted to determine when repeated nucleotides areduplicates; a calculate slopes module adapted to calculate a slope foreach nucleotide position in the base sequence and to display a slopesreport; and a compare sequences module adapted to compare the targetsequence to the base sequence and to display a similarity report.

In another embodiment, the plots module comprises a spectral arraymodule, adapted to plot aligning coefficients for a base sequence and atarget sequence; a single strand module adapted to plot a single strandfor the base sequence and the target sequence; a slopes module adaptedto calculate a slope for each nucleotide position in the base sequenceand to display a plot of the slopes, and an ω_(N) module adapted tocalculate ω_(N) for the base sequence and to display a plot of theω_(N).

Another aspect of the present invention relates to a method for sequenceanalysis. The method comprises the steps of reading a sequence file;selecting a target sequence and a base sequence from said file;performing a non-binary comparison between the target and the basesequences, wherein the non-binary comparison generates a comparisonvalue; and determining a similarity between the target and the basesequences based on the comparison value.

In an embodiment, the method further comprises the steps of writingaligned sequences to the sequence file and calculating an alignmentpercentage.

In another embodiment the method farther comprises the step ofgenerating at least one of a two-dimensional spectral array plot or atwo-dimensional single strand plot.

In another embodiment, the step of performing a non-binary comparisonincludes using a look-up table containing non-binary similarity scorevalues for a plurality of possible comparisons between two sequenceelements.

The foregoing and other features and advantages of the invention will beapparent from the following, more particular description of a preferredembodiment of the invention, as illustrated in the accompanying drawingswherein like reference numbers generally indicate identical,functionally similar, and/or structurally similar elements.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a flow chart of an exemplary method according to thepresent invention.

FIG. 2 depicts an exemplary embodiment of sub-modules of the DNAAnalysis modules according to the present invention.

FIG. 3 depicts an exemplary embodiment of a GUI main window in aVariation Sequence Software Application (hereinafter “VaSSA”).

FIG. 4 depicts an exemplary embodiment of a FILE MENU window in VaSSA.

FIG. 5 depicts an exemplary embodiment of a NOTEBOOK VIEWER window inVaSSA.

FIG. 6 depicts an exemplary embodiment of a SEQUENCE SUMMARY REPORTwindow in VaSSA.

FIG. 7 depicts an exemplary embodiment of a SEQUENCE VIEW REPORT windowin VaSSA (SEQ ID NOS 2-4 disclosed respectively in order of appearance).

FIG. 8 depicts an exemplary embodiment of a SEQUENCE VIEW STATS windowin VaSSA.

FIG. 9 depicts an exemplary embodiment of a ALIGN SEQUENCE menu windowin VaSSA.

FIG. 10 depicts an exemplary embodiment of an ALIGNED SEQUENCE REPORTwindow in VaSSA (SEQ ID NOS 5-6 disclosed respectively in order ofappearance).

FIG. 11 depicts an exemplary embodiment of a QUERY REPEAT window inVaSSA.

FIG. 12 depicts an exemplary embodiment of a QUERY REPEAT REPORT windowin VaSSA (SEQ ID NO 7).

FIG. 13 depicts an exemplary embodiment of an OMEGA SUBZERO window inVaSSA.

FIG. 14 depicts an exemplary embodiment of an OMEGA SUBZERO REPORTwindow in VaSSA.

FIG. 15 depicts an exemplary embodiment of a QUERY OMEGA REPEAT MENUwindow in VaSSA.

FIG. 16 depicts an exemplary embodiment of a QUERY OMEGA REPEAT REPORTin VaSSA (SEQ ID NO 7).

FIG. 17 depicts an exemplary embodiment of a CALCULATE SLOPE window inVaSSA.

FIG. 18 depicts an exemplary embodiment of a CALCULATE SLOPE REPORT inVaSSA (SEQ ID NO 8).

FIG. 19 depicts an exemplary embodiment of a COMPARE SEQUENCE window inVaSSA.

FIG. 20 depicts an exemplary embodiment of a COMPARE SEQUENCE REPORTwindow in VaSSA.

FIG. 21 depicts an exemplary embodiment of a SPECTRAL ARRAY window inVaSSA.

FIG. 22 depicts an exemplary embodiment of a SPECTRAL ARRAY PLOT windowin VaSSA.

FIG. 23 depicts a picture of a SPECTRAL ARRAY FORMULA.

FIG. 24 depicts a schematic drawing of a spectral array formula example.

FIG. 25 depicts a picture of a SPECTRAL ARRAY TRIANGLE STRUCTURE.

FIG. 26 depicts an exemplary embodiment of a SINGLE STRAND window inVaSSA.

FIG. 27 depicts an exemplary embodiments of SINGLE STRAND PLOT REPORTwindows in VaSSA comparing spectral array plots of two 360 basesequences (top) and a region from position 250 to position 295 of thosesequences (bottom), with single base resolution.

FIG. 28 depicts exemplary embodiments of additional SINGLE STRAND PLOTREPORT windows in VaSSA, showing comparisons between single strandsequences.

FIG. 29 depicts an exemplary embodiment of a Plot Slopes window inVaSSA.

FIG. 30 depicts a slopes plot for a single sequence.

FIG. 31 depicts an exemplary embodiment of an OMEGA SUBN window inVaSSA.

FIG. 32 depicts an exemplary embodiment of an OMEGA SUBN PLOT window inVaSSA.

FIG. 33 depicts the chemical structure of the four bases of nucleicacids guanine, cytosine, adenine, and thymine, and uracil, whichreplaces thymine in RNA

FIG. 34A depicts a picture of the different elements involved in A\Gcomparison.

FIG. 34B depicts a picture of the different elements involved in G\Acomparison.

FIG. 34C depicts a picture of the different elements involved in A\Ccomparison.

FIG. 35 depicts an exemplary embodiment of the DNA topological conjugacymodule according to the present invention.

FIG. 36 depicts an exemplary embodiment of the DNA approximate moduleaccording to the present invention.

FIG. 37 depicts an exemplary embodiment of the DNA aligner moduleaccording to the present invention.

FIG. 38 depicts an exemplary embodiment of the non-binary sequencecomparison system according to the present invention.

DETAILED DESCRIPTION

Embodiments of the present invention provide an integrative system foranalyzing and determining sequences' structural behavior over a discretetopology space. The technology provides, among other things, newimproved measurable methods, including normalization, compressiontechnique, structural classification and topological conjugacy methods.These combinations of analytical methods take into account biology,chemistry, and computational mathematical techniques generatingnumerical governing properties, and/or structural behavior patterns ofgenomic data.

The present invention can be used in a wide range of bioinformaticsapplications. The integrative system and method of the present inventionprovide single sequence plots and other data for nucleotide sequences ofessentially any length (e.g., from 50 bases to two million bases). Theintegrative system and method of the present invention are capable ofproviding comparative data for a large number of sequences due toefficient processing steps. For example, the system has beendemonstrated to operate extremely fast with 500 sequences of 500 bases.Comparisons of 1000, 10,000, 100,000, 1,000,000, or more sequences arewithin the scope of the invention.

The system of the present invention uses a non-binary method thatgenerates meaningful comparative information within the homology rangeof 0% (no identity) to 100% (complete identity). The non-binary methodof the present invention is much more discriminating than typical binarycomparisons and can resolve degrees of sequence difference that would beindistinguishable in a binary comparison.

The system and method of the present invention are effective incomparing sequences despite the presence of insertions or deletions ofany length. An alignment module provides both global and localoptimization to permit meaningful comparisons. Single strand plots andcomparisons can be generated in coding (decomposable) regions andnon-coding (indecomposable) regions having chaotic sequences or omegarepeats.

The DNA bases (A, T, G, and C) are used in the description that followsbelow. However, it should be understood that the system and method ofthe present invention are applicable not only to DNA but to allnucleotides, including RNA (substituting Uracil for Thymine), LNA, PNA,and other synthetic nucleotide variants.

The displays shown in the figures typically depict only nucleotidesequences. As should be apparent, for coding regions, the amino acidsequence corresponding to the codons can also be displayed, usingconventional techniques well known to one skilled in the art.

The method of the present invention involves analyzing, retrieving, anddisplaying genomic information. The system and method of the presentinvention provide tools for collecting, storing, analyzing, andretrieving genomic, proteomic, and medical data, data mining and datavisualization and display; sequence alignment and pattern recognition;and structure prediction. For example, the system and method of thepresent invention can be used for predictive biochemical models, insilicon assays, distributed computing, diagnosis, and design of atherapeutic plan.

The system of the present invention is composed of one or more modules.The modules and system of the present invention can be practiced by anindividual operating a stand-alone computer, or as part of a distributedcomputing “system” operated by several individuals. The presentinvention also encompasses various aspects of the system, such as thehardware, software, subsystems, components of the subsystems, andstructures of data produced, compiled, or assembled using the system.Furthermore, the present invention encompasses methods and equipment forgathering, producing, and displaying the relevant data, and associatedanalytical instrumentation, as well as methods of operating and usingthe instrumentation. Business methods of using the system and method ofthe present invention are also contemplated, such as sellingsubscriptions for a sequence analysis tool.

The practice of the embodiments described in further detail below willemploy, unless other wise indicated, conventional methods ofmicrobiology, molecular biology, and immunology within the skill of theart. Such techniques are explained fully in the literature. Allpublications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“VaSSA” refers to Variation Sequence Software Application.

A “computer” refers to any apparatus that is capable of accepting astructured input processing the structured input according to prescribedrules, and producing results of the processing as output. The computercan include, for example, any apparatus that accepts data, processes thedata in accordance with one or more stored software programs, generatesresults, and typically includes input, output, storage, arithmetic,logic, and control units. Examples of a computer include: a computer; ageneral purpose computer; a supercomputer; a mainframe; a supermini-computer; a mini-computer; a workstation; a micro-computer; aserver; an interactive television; a web appliance; a telecommunicationsdevice with internet access; a hybrid combination of a computer and aninteractive television; a portable computer; a personal digitalassistant (PDA); a portable telephone; and application-specific hardwareto emulate a computer and/or software, for example, a programmable gatearray (PGA) or a programmed digital signal processor (DSP). A computercan be stationary or portable. A computer can have a single processor ormultiple processors, which can operate in parallel and/or not inparallel. A computer also refers to two or more computers connectedtogether via a network for transmitting or receiving information betweenthe computers. An example of such a computer includes a distributedcomputer system for processing information via computers linked by anetwork.

A “machine-accessible medium” refers to any storage device used forstoring data accessible by a computer. Examples of a computer-readablemedium include: a magnetic hard disk; a floppy disk; an optical disk,such as a CD-ROM and a DVD; a magnetic tape; a memory chip; and acarrier wave used to carry computer-readable electronic data, such asthose used in transmitting and receiving e-mail or in accessing anetwork.

“Software” refers to prescribed rules to operate a computer. Examples ofsoftware include: software; code segments; instructions; softwareprograms; computer programs; and programmed logic.

A “computer system” refers to a system having a computer, where thecomputer comprises a computer-readable medium embodying software tooperate the computer.

An “information storage device” refers to an article of manufacture usedto store information. An information storage device has different forms,for example, paper form and electronic form. In paper form, theinformation storage device includes paper printed with the information.In electronic form, the information storage device includes acomputer-readable medium storing the information as software, forexample, as data.

The following terms are not in the standard glossary of genetics andbioinformatics.

A “string” is a sequence of characters. A sequence may be considered asa n×1 matrix known as an n-tuple of objects (characters). In the case ofnucleotide sequences, e.g. DNA, RNA, or synthetic or other variants,each nucleotide element has a unique position in the string which is adiscrete set.

Example: AGCAATATAGGA (SEQ ID NO 1) is a string of characters whoselength is 12.

A “subsequence” of a string S means a sequence of characters of S thatneed not be consecutive in S, but do retain their order as given in S.

Example: ACG is a subsequence of ACTCGT.

“f(n)=O(g(n))”: Let f(n) and g(n) be functions. Then f(n)=O(g(n)) if andonly if there is a constant c such that, for all n sufficiently large|ƒ(n)|≦cg(n).

“S₄” is the DNA sequence set on the four Nucleotides: A, C, G, and T.

σ_(L):S₄→S₄ given by σ_(k,L) (s₀s₁s₂ ●θ● s_(n) ●●●)=s₀ s₁s₂ ●●● s_(n)●●● where k=1 (which represents shifting by 1) and the L representsmoving from left to right. Thus σ_(L) is a continuous DNA valuedfunction defined on S₄ . One way to visualize the map is that it simply“forgets” the first entry in a sequence and focuses on all other entriesto the right (i.e., the underlined portion of the sequence above). Theintuitive notion of this DNA continuity can be described by stating thatthe asymptotic linguistic variation above on a small neighborhood of anyposition DNA subsequence in S₄ will vary only slightly from thatposition. This variation can be made as small or as large as one wouldlike it to be by decreasing or increasing the size of neighborhood.

σ_(t,R) is an analog map to the above that is shifting to the left by tunits and reading from the right. The continuity of these maps allowsthe maps to be combined.

Forward and backward orbit of a subsequence: The forward orbit of asubsequence z is the set of points z, σ_(L)(z),σ_(L) ²(z),σ_(L) ³(z),●●● and is denoted by O⁺(z). The backward orbit of a subsequence z isthe set of points z, σ_(R)(z),σ_(R) ²(z),σ_(R) ³(z), ●●●, and is denotedby O⁻(z).

Fixed and periodic subsequence: The DNA subsequence s is a fixedsubsequence for σ_(L) if σ_(L)(s)=s. The DNA subsequence s is a periodicsubsequence of period n, if σ^(n) _(L)(s)=s. The least positive n iscalled the prime period of s. The set of all iterates of a periodicpoint form a periodic orbit.

Eventually periodic: A DNA subsequence s is eventually periodic ofperiod n, if s is not periodic but there exists m>0 such that σ_(L)^(n+i)(s)=σ^(i)(s) for every i≧m. That is σ_(L) ^(i)(s) is periodic fort≧m.

Forward asymptotic: Let s be a DNA subsequence which is periodic ofperiod n. A subsequence x is forward asymptotic to s, if${\lim\limits_{iarrow\infty}{\sigma_{L}^{i\quad n}(s)}} = {s.}$The stable set of s denoted by S^(s)(s) consists of all subsequencesforward asymptotic to s.

“Aligner” is a version of multiple sequence alignment analysis.

“Omega Comparator” is the single and multiple sequence base search baseon the ω_(o) measure.

“Spectral Array” is a series of calculations which allows one to compareall nucleotides in multiple strings which generates its unique structurewith respect to the ω_(o) measure that enables one to find the optimallinguistic behavior.

DNA ω_(o) Genetic Code Viewer” is a finer classification of the geneticcode with the measure ω_(o).

“Stable Analytical Profiler” is a technique that defines a set of allsubsequences forward asymptotic to a target subsequence.

“Unstable Analytical Profiler” is a technique that defines a set of allsubsequences backward asymptotic to a target subsequence.

Chaotic: σ_(L)(z) is said to be chaotic if, (1) σ_(L)(z) has a sensitivedependence with respect to a target subsequence; (2) σ_(L)(z) istopological transitive; and (3) the periodic subsequences are dense withrespect to a string or a data set.

“Symbolic DNA Orbit” is the asymptotic symbolic behavior of a targetsubsequence in a sequence in an iterative process.

“Analytical DNA Orbit” is the asymptotic linguistic behavior of a targetsubsequence in a sequence.

“DNA Approximate Analysis” is a series of techniques which give precisestructural behavior to low complexity subsequences.

“Chaotic Region Classification” is a technique which uniquely partitionssubsequence targets in three categories: (1) targets sensitivelydependent on initial conditions, (2) targets that are topologicallytransitive, and (3) periodic subsequence that are dense in their DNAsequence.

The “DNA Derivative” is a measurement which enables one to observechange qualitatively from one nucleotide to the next in a DNA sequence.

The “DNA Bifurcation” is a technique which observes the change insubsequence under different parameters.

“DNA Topological Conjugate” is a technique which shows when differentmappings of σL(z) are completely equivalent.

“Confidence Score” is a measure which classifies a family of sequencesfrom closest to farthest to a target sequence. The omega similarityscore, or ω_(o) measure, is defined as${{\omega_{0}( {s,t} )} = \frac{\sum\limits_{i = 0}^{N}{s_{i}/t_{i}}}{16*N}},$wherein s_(i)/t_(i) is a non-binary function, examples of which aredefined in Table 1 and 2, and N is the number of nucleotides in theshorter of the two sequences being compared. The omega similarity scoreis a non-binary comparison of any two nucleotide strings, s and t, atbase position i, with the value of the comparison given in a look-uptable.

Embodiments of the present invention are discussed in detail below.While specific exemplary embodiments are discussed, it should beunderstood that this is done for illustration purposes only. A personskilled in the relevant art will recognize that other components andconfigurations can be used without parting from the spirit and scope ofthe invention.

FIG. 1 is an exemplary embodiment. The method 100 of the presentinvention may include the steps of: reading a sequence file (101);selecting a target sequence and a base sequence from the file (103);comparing the target sequence to the base sequence(s) using a non-binarycomparison (105) and generating a similarity score (107); and writingaligned sequences to the file (109). Optionally, the method 100 mayfurther include the steps of generating visual representations of thecomparisons (111), calculating an alignment percentage; and/orgenerating a two-dimensional single strand plot or spectral array plot(113), a multi-strand report (115) or other plot (117).

A sequence file may be a machine-readable file containing one or moregenetic sequences. There are a variety of acceptable formats for DNAsequences. The EMBL format is acceptable. A sequence file in this formatmay contain several sequences. One sequence entry starts with anidentifier line (“ID”), followed by further annotation lines. The startof the sequence may be marked by a line starting “SQ” and the end of thesequence may be marked by two slashes (“//”). FASTA format is alsoacceptable. A sequence in FASTA format begins with a single-linedescription, followed by lines of sequence data. The description linemust begin with a greater-than (“>”) symbol in the first column. Manyother formats such as GCG, GenBank, and IG may also be accepted.

The sequence data may be in text form, e.g., ASCII, or some otherrepresentation readable by a computer executing the method of theinvention. Reading the sequence file may include directly typingsequences in, reading from a disk, or accessing the public domain usinga well-known interface such as Entrez. The files can be stored andanalyzed, or analyzed “on the fly”. The user may choose to read a singlefile or multiple files, or the whole data base, or any subsequence ofany length in a file or multiple files, or the whole data base.

A target is a subsequence of any length. A user may choose to perform ananalysis on a database, or on a file which enables him to observe thestructural behavior. The targets are distinguished from each other intwo steps. The first biological connection is the alphabets that makeupthe subsequence target. The second connection is the omega zerobiological connection.

In one embodiment, the step of generating the spectral array plotcomprises the steps of calculating ω_(N); performing a radialcomparison; extracting alignment coefficients; and plotting thealignment coefficients.

In another embodiment, the step of generating the spectral array plotfurther comprises the steps of reversing one of the base or the target;and reversing a mod.

In another embodiment, the step of performing a non-binary comparisonincludes the step of using a look-up table containing non-binarysimilarity score values for a plurality of possible comparisons betweentwo sequence elements.

In yet another embodiment, the method of the present invention containsthe steps of comparing a molecular structure of a first nucleotide to asecond nucleotide; determining a first non-binary similarity score basedon said comparison; populating a look-up table with the similarityscores for each nucleotide; and using the look-up table to calculate asecond non-binary similarity score that compares a target sequence (t)of nucleotides to a base sequence (s) of nucleotides.

FIG. 38 depicts an embodiment of the non-binary sequence comparisonsystem 10 of the present invention. The system 10 comprises an analysismodule 200, adapted to calculate a non-binary similarity score between afirst nucleotide sequence and a second nucleotide sequence, a filemanagement module 300, a plot module 400 and, optionally, a reportmodule 500, a user options module 600, and/or a user help module 700.

The file management module 300 of the non-binary sequence comparisonsystem 10 manages sequence files. In one embodiment, the file managementmodule 300 comprises a load sequences module 310, adapted to load atleast one sequence file; a flush active sequence module 320, adapted toflush a sequence file from a memory; and a flush loaded sequence module330, adapted to flush a loaded sequence file from the memory. In anotherembodiment, the load sequence module 310 comprises a loaded sequencedisplay module 312, adapted to generate and display a summary reportnotebook page when a sequence is loaded. The summary report notebookpage is adapted to display a sequence file name and a number ofsequences.

In another embodiment, the plots module 400 of the non-binary sequencecomparison system 10 comprises a spectral array module 410, adapted toplot aligning coefficients for a base sequence and a target sequence; asingle strand module 420 adapted to plot a single strand for the basesequence and the target sequence; a slopes module 430 adapted tocalculate a slope for each nucleotide position in the base sequence andto display a plot of the slopes, and an ω_(N) module 440 adapted tocalculate ω_(N) for the base sequence and to display a plot of theω_(N). In a preferred embodiment, the spectral array module 410 isfurther adapted to calculating an ω_(N) value for radial compare andextracting aligning coefficients. In another preferred embodiment, thesingle strand module 420 is adapted to calculate an ω_(N) value for thebase sequence and the target sequence.

In another embodiment, the report module 500 of the non-binary sequencecomparison system 10 of the present invention is adapted to generate anddisplay a sequence summary, a listing of the contents of each loadedsequence, and/or statistical information about each loaded sequence.

In yet another embodiment, the analysis module 200 of the non-binarysequence comparison system 10 comprises an align sequences module 201,adapted to align a target sequence to a base sequence and to display analignment report; an ω₀ module 203, adapted to calculate an ω₀ score fora sequence and to display the ω₀ score; a query repeat module 205,adapted to locate multiple occurrences of the target sequence in thebase sequence and to display the multiple occurrences; a query omegarepeats module 207, adapted to determine when repeated nucleotides areduplicates; a calculate slopes module 209, adapted to calculate a slopefor each nucleotide position in the base sequence and to display aslopes report; and a compare sequences module 211, adapted to comparethe target sequence to the base sequence and to display a similarityreport.

In a preferred embodiment, the align sequences module 201 is furtheradapted to perform the action of reversing said base sequence, reversinga mod, aligning the base and the target to a shortest length,calculating an alignment percentage, and/or calculating an omegasimilarity score.

In another preferred embodiment, the compare sequences module 211 isfurther adapted to perform the action of reversing the base sequence,reversing the target sequence, reversing a mod, calculating an ω_(N)value for each of the base and the target sequences, converting the baseand the target sequences to binary, calculating a distance between thebase sequence and the target sequence, and determining if the distanceexceeds a bound.

FIG. 2 depicts a layout of a preferred module decomposition of the DNAanalysis portion of the VaSSA architecture. The modules in thedecomposition are discussed in more detail below. Submodules aredepicted in flowchart form in FIGS. 35 to 38.

Module Decomposition of VaSSA Architecture

DNA Analysis Module groups 200

-   -   SSDA (Single Strand DNA Analysis) module group 210    -   MSDA (Multi-Strand DNA Analysis) module group 240

SSDA (Single Strand DNA Analysis) (FIG. 2)

-   -   DNA Approximate Module 212    -   Chaotic Region Classification Module 214    -   The DNA Derivative Module 216    -   The DNA Bifurcation Module 218    -   DNA Orbit Module 220    -   Analytical Behavior Profiler Module 222    -   DNA Topological Conjugacy Module 224    -   Structural Stable Region Module 226    -   Indecomposable Region Module 228    -   DNA Complexity Bases Module 230    -   DNA Aligner Module 232

MSDA (Multi-Strand DNA Analysis) (FIG. 2)

-   -   DNA Approximate Module 242    -   Chaotic Region Classification Module 244    -   The DNA Derivative Module 246    -   The DNA Bifurcation Module 248    -   DNA Orbit Module 250    -   Analytical Behavior Profiler Module 252    -   DNA Topological Conjugacy Module 254    -   Structural Stable Region Module 256    -   Indecomposable Region Module 258    -   DNA Complexity Bases Module 260    -   DNA Aligner Module 262

DNA Topological Conjugacy Module 224 and 254 (FIG. 35)

-   -   a. Analytical Profiler Module 3501    -   b. Analytical Mapper Module (Creation of Analytical Mapping)        3503    -   c. Conjugacy Comparison Module 3505    -   d. First Iteration Analysis Module 3507    -   e. Phase Portrait Generator Module 3511

DNA Approximate Module 212 and 242 (FIG. 36)

-   -   a. Holomorphic Form Generator Module 3601    -   b. Approximate Constructor Module 3603    -   c. P & Q Coefficient Calculator Module 3605    -   d. JC-DNA Curve Generator Module 3607    -   e. Low Complexity Generator Module 3609    -   f. Target Classifier Module 3611    -   g. Symbolic DNA Orbit Module (also a child of SSDA and MSDA)        3613    -   h. Analytical DNA Orbit Module (also a child of SSA and MSDA)        3615

DNA Orbit 220 and 250 (Analytical DNA Orbit Module)

-   -   Symbolic DNA Orbit Module 3701        -   a. Symbolic Flow Generator Module 3703        -   b. Row Difference Generator Module 3705        -   c. Orbit Generator Module 3707    -   Analytical DNA Orbit Module 3709        -   a. Analytical Forward Profiler Module 3711        -   b. Analytical Backward Profiler Module 3713        -   c. DNA Attractor Generator Module 3715        -   d. DNA Repeller Generator Module 3717

Chaotic Region Classification Module 214 and 244

-   -   Chaotic Region Classifier 3801        -   a. DNA Sensitivity Generator Module 3803        -   b. DNA Transitivity Generator Module 3805        -   c. Dense Periodic Sequence Generator Module 3807

The DNA Bifurcation Module 218 and 248

-   -   Splitter Classifier 3901        -   a. DNA Transitivity Splitter Profiler Module 3903        -   b. DNA Dense Splitter Profiler Module 3905

The DNA Derivative Module 216 and 246

-   -   Derivative Generator Module 4001    -   Monotonic Generator Module 4003        -   a. Positive Measure Module 4005        -   b. Negative Measure Module 4007

Analytical Behavior Profiler Module 222 and 252

-   -   DNA Approximate Module 4101    -   Chaotic Region Classification Module 4103    -   The DNA Derivative Module 4105    -   The DNA Bifurcation Module 4107    -   DNA Orbit Module 4109    -   Analytical Behavior Profiler Module 4111    -   DNA Topological Conjugacy Module 4113    -   Structural Stable Region Module 4115    -   Indecomposable Region Module 4117    -   DNA Complexity Bases Module 4119    -   DNA Aligner Module 4121    -   Algebraic Structure Generator Module 4123        -   a. Group Generator Module 4125        -   b. Semi-Group Generator Module 4127        -   c. Ring Generator Module 4129        -   d. Analytical Set Generator Module 4131    -   Homomorphism-Generator Module 4133    -   Isomorphism-Generator Module 4135

Structural Stable Region Module 226 and 256

-   -   Repeat Generator Module 4201    -   Forward Asymptotic Module 4203    -   Stability Profiler Module 4205

Indecomposable Region Module 228 and 258

-   -   DNA Orbit Analysis Module 4301    -   Non-repeat Generator Module 4303    -   Indecomposable Profiler Module 4305

DNA Complexity Bases Module 230 and 260

-   -   Repeat Generator Module 4401    -   Universal DNA Basis Generator Module 4403    -   Density Generator Module 4405

DNA Aligner Module 232 and 262

-   -   Symbolic Aligner Module 4501        -   a. Single Strand Generator Module 4503        -   b. Multi-Single Strand Generator Module 4505    -   Omega Comparison Aligner Module 4507        -   a. Omega Single Strand Generator Module 4509        -   b. Multi-Single Strand Generator Module 4511            Descriptions of Main Modules of VaSSA

DNA Approximate Module 212 or 242: This module reduces the polynomialtype construction that is in VaSSA. It shows that not all thecoefficients off are needed to perform a calculation. Also, theapproximant generates data that can be used for visualization of thelinguistic structure behavior of low complexity subsequences. Thisprocedure is performed without losing any biological information. Theapproximant is at a lesser order which provides a faster, more preciseanalysis and the calculation gives a better fitting of the originalfunction.

Chaotic Region Classification Module 214 or 244: This module possessesthree ingredients: unpredictability, elements of regularity, andelements that cannot be broken down to smaller subsequences.

DNA Derivative Module 216 or 246: This module creates an environmentwhere monotonic changes in content can be observed as a DNA string isread from left to right and/or from right to left. When the DNAderivative is positive, the information being transferred is increasing.When DNA derivative is negative, the information being transferred isdecreasing. When the DNA derivative is zero, the information beingtransferred is constant.

DNA Bifurcation Module 218 or 248: This module analyzes the changes inthe DNA maps as they undergo parameter changes. These changes ofteninvolve the periodic subsequences of DNA but also involve other changesas well.

DNA Orbit Module 220 or 250: Even though analysis of DNA sequences ismathematical in nature, this module creates an environment which answersthe somewhat nonmathematical question: where do subsequences go and whatdo they do when they get there? This module connotes the geometricprocess of taking one subsequence to another assuming that DNA sequencesare discrete sets.

Analytical Behavior Profiler Module 222 or 252: This module takes intoaccount all of its children modules and then connects them throughalgebraic functional methods which does not lose the content of thebiology. It then further refines information by dissecting the dynamicinformation from the child modules to algebraic equivalence classes.

DNA Topological Conjugacy Module 224 or 254: This module relates datasets to data sets, DNA sequences to DNA sequences, and multiple DNAsequences to DNA sequences. It creates an environment which classifiessequences that are completely equivalent and not equivalent.

Structural Stable Region Module 226 or 256: This module relates tounderstanding all orbits, and to identifying the set of orbits which areperiodic, eventually periodic asymptotic, etc. Implementation ofqualitative and/or geometric techniques to understand a given data set.

Indecomposable Region Module 228 or 258: This module relates tounderstanding all non-orbits, and to identify the set of non-orbitswhich are not periodic, eventually periodic or asymptotic, etc.Implementation of qualitative and/or geometric techniques to understanda given data set.

DNA Complexity Bases Module 230 or 260: This module creates a universalDNA set in which observations of how non-periodic subsequences arearbitrarily close to another sequence can be made. The module creates anenvironment where linguistic behavior agrees in a large number ofplaces, which create linguistically dense orbits. These orbits arecalled topologically transitive.

DNA Aligner Module 232 or 262: This module is VaSSA's version of asystem of tool kits analyzing sequence alignment. In addition, themodule may be enhanced with additional biological information modulessuch as symbolic DNA orbit, etc.

FIG. 3-FIG. 28 depict exemplary embodiments of a graphical userinterface (GUI) with the VaSSA, during VaSSA execution.

The aligned sequences may then be written back to the sequence file, ora different file. The percentage of alignment may then be calculated,which shows the percentage of the two sequences that are in alignment.

An omega similarity score (which is ω_(o)) may also be calculated. Thealgebraic structure of ω_(o) is defined as${\omega_{0}( {s,t} )} = {\frac{\sum\limits_{i = 0}^{N}{s_{i}/t_{i}}}{16*N}.}$The omega similarity score, or ω_(o) measure, is a non-binary comparisonof any two nucleotide strings, s and t. This can easily be modified foranalysis on a single string by substituting s_(i)/t_(i+1) fors_(i)/t_(i) in the foregoing equation.

The omega similarity score may be calculated in several ways. The valueof the s_(i)/t_(i) comparison is based on the chemical structure of thenucleotides of DNA. In DNA, there are four possible bases: adenine (A),cytosine (C),guanine (G), and thymine (T). In RNA, the thymine isreplaced by uracil (U). The structure of these bases is shown in FIG.33. The purines, adenine and guanine, have a two ring structure, and thepyrimidines, cytosine, thymine and uracil, have a single ring structure.The value s_(i)/t_(i) represents the differences in structure betweenthe various bases. In the purine base structure, there are two rings,which can be considered the large, six-membered ring and the small,five-membered ring. The pyrimidine structures have only one ring. Themeasurement can be broken down into four categories: purine\purine,pyrimidine\pyrimidine, purine\pyrimidine and pyrimidine\purine.

Traditional methods of comparing DNA sequences operate by comparing thebase sequences in a binary fashion, i.e., simply assessing whether thebase is the same or different. In one aspect, the present invention is amethod of comparing DNA sequences that takes into account not only thatbases are different, but measures the magnitude of the difference. Thus,the invention includes a non-binary method of comparing DNA sequences.

In a first embodiment, steric considerations are primarily considered.In this embodiment, a value of 0 is assigned if the bases are identical,1 is assigned for purine\purine, pyrimidine\pyrimidine arrangements,i.e. where the bases are the different but the ring size is unchanged,and 2 is assigned for purine\pyrimidine and pyrimidine\purine, where thering size of the base changes. Thus, ω_(o) reflects not only adifference in the identity of the base, but also the degree ofdifferences between the chemical structure of the purines and thepyrimidines.

The first embodiment is illustrated in Table 1: TABLE 1 S s/t A G C T TA 0 1 2 2 G 1 0 2 2 C 2 2 0 1 T 2 2 1 0

A second embodiment of the invention further considers the number ofelements in the base s_(i) not present in the base t_(i) in therespective position of the molecular structure. A purine\purinemeasurement compares both the large ring and the small ring. This iswhere the molecular arrangement is most similar and both purinemolecules behave similarly with respect to size and arrangement of theirchemical elements. The measurement, referred to herein as ω₀, iscalculated in one embodiment by counting the number of atoms present inthe first sequence that are not present in the second sequence. Forexample, if a first sequence s has a guanine (“G”) nucleotide atposition i and the second sequence t has an adenine (“A”) nucleotide atthe corresponding position, there then ω₀ measure at position i(referred to herein as s_(i)/t_(i)) is calculated by determining thenumber of atoms in si not present and/or in a different position int_(i). Referring now to FIG. 33, in the guanine molecule, the oxygenatom (1), the hydrogen atom (2) and the NH2 group of atoms (3, 4, 5)bonded to the large ring, and the hydrogen (6) and carbon (7) atoms inthe small ring opposite the double bonded carbon atoms are either notpresent or in a different position in the adenine molecule. Accordingly,s_(i)/t_(i)=7 where s_(i)=G and t_(i)=A. Thus, ω₀ reflects the degree ofdifferences and similarities in chemical structure of the purines. It isassumed that these differences and similarities have biologicalsignificance in coding and non-coding regions of the nucleotidesequence. The calculation of ω₀ may be modified with more preciseinformation at the bonding level for each chemical element in otherembodiments.

In the calculation of the omega measure, when the omega measure isidentically zero, the chemistry is identically the same. Where the omegameasure is not identically zero, the omega measure gives a number whichrepresents the number of different chemical elements. A completeanalysis on the four nucleotides is displayed in the Table 2 below. Thes_(i)/t_(i) value in a pyrimidine\pyrimidine analysis is carried out inan analogous fashion as the purine\purine measure, except only thesingle ring is considered. In a purine\pyrimidine or pyrimidine\purinemeasurement, the large ring of the purine is compared to the ring of thepyrimidine but the comparison is performed counterclockwise on the largering of the purine and clockwise on the pyrimidine ring (or vice-versa).The structures of the molecules are shown in FIG. 33. However, themeasure value does not change since the structure of the nucleotideelements structure does with respect to two ring verses one ring, etc.

Using this second embodiment of the invention, a matrix can be generatedto determine values of s_(i)/t_(i), as seen in Table 2: TABLE 2 S s/t AG C T T A 0 7 4 9 G 6 0 7 7 C 6 10 0 6 T 9 8 4 0

FIGS. 34A-34C display some examples of the result of the omega count andthe chemical elements involved. The figures demonstrate graphically whyA/G is more similar than A/C and A/T, and G/A is more similar than G/Cand G/T, and so on. Even though the omega measure generates numbers forG/A and G/T that are the same, the chemical elements involved aredifferent. The redundancy of the elements of the table is clarified bythe figures, which depict the elements involved. The real-worldsignificance of these similarities or differences is to be able todescribe how similar or how different a set of sequences is, withoutlosing the integrity of traditional biological relevance in presentsequence alignment searches. Other difference matrices can be used basedon other chemical comparisons between the bases.

In view of the present disclosure, persons skilled in the art will beable to construct corresponding tables for RNA and protein

In one embodiment, two alternative sequences t and r:

-   -   t=AAGCC    -   r=AAGAC    -   are compared to a native sequence s:    -   s=ATAGC

It is observed that r and t differ from s by three bases. However, r ands are not identical, and the question to be considered is: which of rand t is more similar to s?

Using a traditional approach, one can define a quantity S(s,t) andS(s,r) to compare t and r, respectively, to s. Using the common BLASTsystem, wherein S(x_(i), y_(j))=s(x_(i)y_(j))={+1, x_(i)=y_(j), −μ,x_(i)≠y_(j) and${{S( {x,y} )} = {\sum\limits_{i,j}^{n,m}{s( {x_{i},y_{j}} )}}},$where μ is a constant,the similarity scores for s and t are:

-   -   S(s,t)=2-3μ    -   S(s,r)=2-3μ    -   No apparent difference is observed.

Using the first embodiment of the invention as described above inconnection with Table 1, values of ω₀(s,r) and ω₀(s,t) are determined asfollows:ω₀(s,r)=(0+2+1+1+0)=4ω₀(s,t)=(0+2+1+2+0)=5.

-   -   Thus, we see that there is a difference.

Using the second embodiment of the invention as described above, valuesof ω₀(s,r) and ω₀(s,t) are determined using (wherein N represents thelength of the shorter of the two sequences being compared):$\begin{matrix}{{\omega_{0}( {s,t} )} = \frac{\sum\limits_{i = 0}^{N}{s_{i}/t_{i}}}{16*N}} & (1)\end{matrix}$as follows:${\omega_{0}( {s,r} )} = {\frac{( {0 + 9 + 6 + 7 + 0} )}{80} = {{22/80} = 0.275}}$${\omega_{0}( {s,t} )} = {\frac{( {0 + 9 + 6 + 10 + 0} )}{80} = {{25/80} = 0.3125}}$Segment r is more similar to s than is t.

Because of the redundancy of the integers in the second embodiment, itis possible to come up with sequences that have the same value forexample A/G verses A/C., however looking at the chemistry involved inthe count are very different. This is an indication of how molecules arecommunicating differently and therefore not transferring the sameinformation.

For sequences of an entire genome, a normalization technique is used andit is presented in equation (2) below. Thus, in a DNA sequence eachposition of a nucleotide represents a unique address in the string. Inshort strands, the denominator is used to measure the intensity of thedifference. For longer strands, the normalization technique discussedbelow in connection with equation (2) is used in which purges theexponential growth of the denominator out. This allows VaSSA to ploteach position with respect to its unique address. The omega measure withrespect to these unique positions generates unique structural behaviorwith respect to each nucleotide as well as how it is profiled withrespect to the strand it is in.

Computer Program Product

In an exemplary embodiment, the method of the present invention may beembodied on a machine-readable medium, that when read by the machinecauses the machine, for example, a computer, to perform the methodsdescribed above. In addition, this embodiment of the invention mayprovide a graphical user interface (GUI) that allows a user to comparesequences of genetic material, and further analyze the sequences and thecomparison results.

For example, as seen in FIG. 3, the GUI may provide modules for filemanagement, reporting, analysis, plotting, setting user options, anduser help.

As shown in FIG. 4, the file management module 300 may further include amodule to load sequences, which may load one or more sequence files. Afile may contain a single sequence or multiple sequences. Thesesequences can be read off a disk, CD, etc. These sequences does not haveto be stored, they could be analyzed “on the fly” as they are received.The sequence files may be FASTA formatted, or any other format. Whenloaded, each sequence may be assigned a unique reference number, and maybe checked to ensure that all characters are valid.

The file management module 300 may also include a module to flush activesequences, which may remove, or “flush”, an active sequence file frommemory. When flushed, the reference numbers for the sequence arepreserved. The file management module 300 may also include a module toflush a loaded sequence from memory. An active sequence is a sequence inwhich analysis is being carried out on while a loaded sequence is asequence also in memory but at the present time there is no analysisbeing done on it.

The module to load sequences may include a module to display a loadedsequence, which may generate and display a summary report notebook pagewhen a sequence is loaded. As shown in FIG. 5, the summary reportnotebook page may display a sequence file name and a number ofsequences.

The report module 500 may generate and display a sequence summary of allloaded sequences including the unique reference number, the sequenceheader, and the sequence length (FIG. 6); a listing of the contents ofeach loaded sequence including the unique reference number and thesequence contents in FASTA format (FIG. 7); and/or statisticalinformation about each loaded sequence including the unique referencenumber, the sequence header, and a count of each standard sequencecharacter (FIG. 8). If a sequence character is not recognized, thereporting module generates a error signal which is listed in an “Error”column in the statistical information about each loaded sequence (FIG.8).

The analysis module 200 may include a number of sub-modules. Forexample, an align sequences sub-module may align a target sequence to abase sequence and display an alignment report (FIG. 9). The alignsequences module may also reverse the base sequence, reverse a mode,align the base and the target to a shortest length, calculate analignment percentage, or calculate an omega similarity score (FIG. 10).The omega similarity score may be used to determine whether and to whatextent the target is similar to the base. If the omega similarity scorevalue is less than ½^(n) where n is the maximum length of the twosequences s and t, the two sequences may be said to be similar. If theomega similarity score value is greater than ½^(n), then the sequencesare said to be dissimilar.

The tasks of the menu options in the VaSSA analysis menu include but notlimited to:

1. Reverse Base

Under the analysis menu of VaSSA, is a reverse base option. One functionof the Reverse base is to enable the user to change the sequence around.For example if the sequence is 5′ to 3′ direction then reverse basefunction reads from the 3′ to 5′ direction (however not the complementstrand direction).

2. Reverse Mod

The function of the Reverse Mod option is to enable one to reverse themod calculations. “Reversing the mod calculations” means changings_(i/t) _(i) to t_(i)/s_(i). This is significant since by definition ω₀is not a symmetrical operation.

3. Align Base and Target Sequences to the Shortest Length

The base and target are two sequence strings of different lengths or thesame length. If the strings are of different lengths then the first partof the analysis is to align and stop at the end of the shortestsequence. If they are the same length, the sequence analysis is carriedout to the end of each string.

4. Calculate Alpha Numeric Alignment Percentage and Omega SimilarityScore

The alpha numeric alignment is an alignment which gives a percentagewhich is the total number of nucleotides aligned over the total numberof nucleotides. As shown in FIG. 13, an omega sub-zero (ω₀) module maycalculate an ω₀ score for a sequence and display the ω₀ score. One base,or all loaded sequences may be chosen. The report can be sorted byreference number, length, or Omega score (FIG. 14). The base sequenceand the mod may each be reversed.

The ω₀ value can also be calculated by the single strand module for thebase sequence and the target sequence. Consider the following singlestrand equation, which is a simplified version of equation 6 (themultiple strand form of the equation will be discussed below):$\begin{matrix}{{{C_{l}( z_{1} )} = {\sum\limits_{\lambda_{1} = l}{c_{\lambda_{1}}z_{1}^{\lambda_{1}}}}},{l = 0},1,2,\ldots} & (2)\end{matrix}$where

z₁ represents a single strand. That is, z₁=s₀s₁ ◯◯◯ s_(k) ◯◯◯ where eachs_(k) is an A,G,C or T.

z₁ ^(λ) ^(i) corresponds to the nucleotide in the λ_(i) th position andλ_(i+1) th position where i is a number in the index set 1=1,2,3, . . ..

The coefficients c_(λ) _(i) =s_(i)/s_(i+1) for the λ ith position andi+1 th position where i is a number in the index set 1=1,2,3, . . . .

Thus, for an exemplary four nucleotide strand z_(i)=ACGT, C₁(z₁) is anarray of coefficients [c₀, c₁, c₂], where each coefficient is calculatedby determining z₁ ^(λ) ^(i) /z₁ ^(λ) ^(i+1) for position i in the strand(except for the last position), which is equal to [A/C, C/G,G/T]=[6,7,8] in this case. These coefficients can be used to form asingle strand plot for strand z₁ in which the position in the strand (inother words, the value of 1) is represented on the x axis and the valueof the corresponding coefficient is represented on the y axis (anexample of single strand plots for two strands is shown in FIG. 27).

A query repeats module may locate multiple occurrences of auser-specified target sequence in the base sequence and display themultiple occurrences. Multiple occurrences of a target sequence arereferred to herein as repeats. VaSSA has two types of repeats: Repeatsand Omega repeats. The repeats are just using the shift function onsymbols and the Omega repeats use the shift function on the measurementof omega similarity. As shown in FIG. 11, The user may select a basesequence to search, and a target sequence to search for. The user mayspecify a threshold to relax or tighten the search. The base or targetsequences may also be reversed. The query repeat module may thengenerate sub-targets when the user specifies a threshold and identifypositions in the base where the target or sub-target appear. In oneembodiment, if the target is AGCT, the query repeat module may generatesub-targets of AGC and GCT. As shown in FIG. 12, the repeat target andsubtargets are identified at the top of the GUI window page along withthe number of times the repeat target and subtargets are detected.Occurrences of the target sequence are identified with hat symbols 1201and occurrences of sub-target sequences are identified with asterisksymbols 1202.

As shown in FIGS. 15 and 16, a query omega repeats module obtainseverything aforementioned with respect to the query repeat module.However, in addition, it picks up how repeated nucleotides in a segmentof a string may be communicating differently (at least with respect tothe omega measure) in another segment of the string. Thus query omegarepeats can pick up when repeats are duplicates and when they are not.

As shown in FIGS. 17 and 18, a calculate slopes module may calculate aslope for each nucleotide position in a base sequence and display aslopes report. In an exemplary embodiment, the slopes may be calculatedusing the following:Ω_(k)=s_(k)/s_(k−1)−s_(k−1)/s_(k)   (3)

where k represents the unique position of a nucleotide in a DNAsequence. ω_(k)=s_(k)/s_(k+1), ω_(k) is the k^(th) term in the ω₀series. The equation may be used to generate information on curvature inthe 2-D profiles. When Ω_(k) is positive, the information beingtransferred is increasing and the bonds that connect the double strandare longer (and thus have a tendency to be weaker than shorter ones).When Ω_(k) is negative, the information being transferred is decreasingand the bonds are shorter connecting the double helix (and have atendency to be stronger). Thus, in a plot of the positives and negativesis a profile of information flows from one position to the next in asequence. The slope graph is a plot of the change information flow. Itshows where information change is the same in the sequence (with zerosin sign chart) and different. It also shows where information is exactlythe same but in the opposite direction. To generate the graph, (anexample of which is shown in FIG. 30) the position of the nucleotide isplotted against the value of the slope. Thus, equation 3 is whatgenerates the sign charts and the slope plots in VaSSA. In both cases,the nucleotide unique position in a strand corresponds to the x-axis andthe value of Ω_(k) corresponds to the y-axis.

In one embodiment, in a sequence AGC, the change from A to G would becalculated as follows: A is at position k-1, G is at k, and C is at k+1.Omega(k), based on the values in Table 2, is then G/C−A/G=10−6=4. Thechange from A to G is therefore positive, and may be represented by a“+” in the slopes report.

As shown in FIGS. 19 and 20, a compare sequences sub-module may comparethe target sequence to the base sequence and display a similarityreport. The compare sequences sub-module may also reverse the basesequence, reverse the target sequence, reverse a mode, calculate anω_(n) value for each of the base and target sequences, convert the baseand the target sequences to binary, calculate a distance between thebase sequence and the target sequence, and determine if the distanceexceeds a bound.

As shown in FIGS. 21-25, the plots module may include a number ofplotting sub-modules. For example, a spectral array sub-module may plotaligning coefficients for a base sequence and a target sequence. Thespectral array sub-module may also calculate an ω_(n) value for radialcompare, and extract aligning coefficients. In a radial comparison, thespectral array sub-module may use the formulas: $\begin{matrix}{{{f( \overset{arrow}{z} )} = {\sum\limits_{l = 0}^{\infty}{C_{l}( \overset{arrow}{z} )}}}{where}} & (4) \\{{{c_{l}( \overset{arrow}{z} )} = {\sum\limits_{\lambda_{1} + \lambda_{2} + \ldots + \lambda_{n = 1}}{c_{\lambda_{1}\lambda_{2}\quad\ldots\quad\lambda_{n}}z_{1}^{\lambda_{1}}z_{2}^{\lambda_{2}}\quad\ldots\quad z_{n}^{\lambda_{n\quad 1}}}}},{l = 0},1,2,\ldots} & (5)\end{matrix}$This formula is for multiple sequences. It allows the generation of aunique spectral analysis is a notation that is used for multiple sumswith respect to 1. These are the coefficients generated in each sequencewith respect to ω₀ to their positions. The nucleotide in each sequenceposition is denoted by Z₁ ^(λ) ¹ Z₂ ^(λ) ²¹ ●●● Z_(n) ^(λn1).

The formation of equations 4 and 5 allows the generation of the plots inVaSSA. The Coefficient Structure of the formula can be captured in atriangle structure which is presented in FIG. 25. The spectral structureis triangle allows to observe optimalization without inserting ordeleting spaces in strands of DNA. FIG. 24 demonstrates with two strandsof how the coefficients are being generated when the formula was used.The single strand plot has the same structure but different values.Because of the non-binary measure, it can be precisely observed thatwhere the plots are equivalent and where they are different. It can alsobe observed that where there is periodicity. Since the function isanalytical, it can be formulated shifts without effecting the uniquenessof nucleotide location. One embodiment is shown in FIG. 27. The spectralarray plot in VaSSA uses the coefficients right down the center of thetriangle structure on FIG. 25. An example of this plot is FIG. 22. Thishas information where they have direct alignment because is the graph iszero their. There are also spikes with a certain heights. Similarinformation can be observe as single strand plot. But the magnitude ofthe difference can be visualized here with respect to the height of thespikes. Also with pointers in the triangles we can a complete phaseportrait which is a different way to do optimization.

As shown in FIGS. 26-28, a single strand sub-module may plot a singlestrand for the base sequence and the target sequence. The single strandsub-module may also calculate an ω_(n) value for the base sequence andthe target sequence. The single strand sub-module may plot usingequation (4), where $\begin{matrix}{{{C_{l}( \overset{arrow}{z} )} = {\sum\limits_{\lambda_{11 = l}}{c\quad\lambda_{1}z_{1}^{\lambda_{1}}}}},{l = 0},1,2,\ldots} & (6)\end{matrix}$is a simplified version of equation (5). However this equation allowsone to profile a single strand.

As shown in FIGS. 29-30, a slopes module may calculate a slope for eachnucleotide position in the base sequence and display a plot of theslopes. A ω_(n) module may calculate ω_(n) for the base sequence anddisplay a plot of ω_(n). The ω_(n) module may use equation (6).

The generate plot of slope will generate the plot on FIG. 30. The slopeplot is a graph of the montonicity of information flow. This plot allowsa user to determine local and global max, and min position on singlestrand plots. It also allows a user to determine concavities in localareas as well as global areas of the single strand plot.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. Thus, the breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should instead be defined only in accordancewith the following claims and their equivalents.

1. A system for sequence analysis, comprising: an analysis module,adapted to calculate a non-binary similarity score between a firstnucleotide sequence and a second nucleotide sequence; and an output incommunication with the analysis module for outputting the similarityscore, wherein said non-binary similarity score is calculated by pairingnucleotides of said first nucleotide sequence with nucleotides of saidsecond nucleotide sequence, obtaining a similarity score for each basepair between said first nucleotide sequence and said second nucleotidesequence, and combining similarity scores of all base pairs between saidfirst nucleotide sequence and said second nucleotide sequence, whereinthe similarity score for a base pair is a non-binary score that dependsupon a similarity of chemical structure between the nucleotides of basepair.
 2. The system of claim 1, wherein the similarity score for a basepair is a first value if the nucleotides in said base pair are the same,a second value if the nucleotides in said base pair are different butare both purines or pyrimidines, or a third value if one nucleotide insaid base pair is a purine and another nucleotide in said base pair is apyrimidine, and wherein the first, second and third values are differentfrom each other.
 3. The system of claim 2, wherein the second value orthe third value is further modified by a degree of differences betweenthe chemical structure of the nucleotides in the base pair.
 4. Thesystem of claim 1, further comprising a graphical user interface (GUI)that comprises a report module adapted to provide reports on loadedsequences; a file management module adapted to manage sequence files;and a plot module adapted to provide sequence analysis plots.
 5. Thesystem of claim 4, wherein the GUI further comprising a user optionsmodule adapted to provide user preferences.
 6. The system of claim 4,wherein said file management module comprises: a load sequence module,adapted to load at least one sequence file; a flush active sequencemodule, adapted to flush a sequence file from a memory; and a flushloaded sequence module, adapted to flush a loaded sequence file fromsaid memory.
 7. The system of claim 6, wherein said load sequence modulecomprises: a loaded sequence display module, adapted to generate anddisplay a summary report notebook page when a sequence is loaded,wherein said summary report notebook page is adapted to display asequence file name and a number of sequences.
 8. The system of claim 4,wherein said report module is adapted to generate and display at leastone of: a sequence summary; a listing of the contents of each loadedsequence, or statistical information about each loaded sequence.
 9. Thesystem of claim 1, wherein said analysis module comprises asingle-strand DNA analysis module and a multi-strand DNA analysismodule.
 10. The system of claim 9, wherein each of said single-strandDNA analysis module and said multi-strand DNA analysis module comprisesat least one module selected from the group consisting of a DNAapproximate module, a chaotic region classification module, a DNAderivative module, a DNA bifurcation module, a DNA orbit module,analytical behavior profiler module, a DNA topological conjugacy module,a structural stable region module, an indecomposable region module, aDNA complexity bases module, and a DNA aligner module.